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Chapter 2 Molecular mechanism of co-translational membrane protein recognition and targeting by SecA recognition and targeting by SecA
2.2 Introduction
Chapter 2 Molecular mechanism of co-translational membrane protein
widely used by cells is to recruit molecular chaperones when the TMD on a nascent polypeptide emerges from the ribosomal exit tunnel. These chaperones protect the nascent TMD from aggregation and also act as or in collaboration with dedicated targeting machinery to co-
translationally deliver the nascent membrane protein to the translocation machinery on the target membrane(Nyathi et al., 2013).
Diverse membrane protein targeting pathways have been discovered. The most well-studied route is mediated by the signal recognition particle (SRP) (Zhang et al., 2010; Zhang and Shan, 2014), which is co-translationally recruited to ribosomes (Chartron et al., 2016; Schibich et al., 2016) and shields TMDs or hydrophobic signal sequences adjacent to the N-terminus of the nascent protein (Jomaa et al., 2016). The interaction of SRP with its membrane receptor SR delivers the ribosome•nascent chain complex (RNC) to the Sec61p translocase at the eukaryotic endoplasmic reticulum (ER), or the SecYEG translocase at the bacterial plasma membrane (Jomaa et al., 2016; Jomaa et al., 2017). In eukaryotic cells, the SRP-independent targeting (SND) components help in the delivery and insertion of a subclass of membrane proteins
harboring internal TMDs to the ER, possibly before the nascent protein finishes its synthesis (Ast et al., 2013; Aviram et al., 2016). The ER membrane protein complex (EMC) could insert a subset of nascent TMDs into the membrane both co- and post-translationally (Chitwood et al., 2018; Guna et al., 2018; Shurtleff et al., 2018). The diversity of membrane protein targeting and translocation machineries are suggested to accommodate different properties of the nascent membrane proteome, such as hydrophobicity (Ast et al., 2013; Guna et al., 2018; Shurtleff et al., 2018), location (Aviram et al., 2016), or topology of the TMDs (Ast et al., 2013; Chitwood et al., 2018; Shurtleff et al., 2018).
SecA is another emerging bacterial protein biogenesis factor that can mediate the co-translational targeting and translocation of some of the membrane proteins (Huber et al., 2011; Singh et al., 2014; Wang et al., 2017). SecA binds to the ribosome near uL23 in proximity to the exit tunnel (Huber et al., 2011; Singh et al., 2014), and could be recruited to many membrane proteins during translation (Huber et al., 2016). The most well-characterized membrane protein substrate for co-translational delivery by SecA is RodZ (Rawat et al., 2015; Wang et al., 2017), a single pass type II membrane protein essential for cell division. SecA is necessary and sufficient for the
targeting of RodZ to the SecYEG translocon in a strictly co-translational mechanism in vitro and vivo (Wang et al., 2017). SecA binds to RNCs bearing the RodZ nascent chain with high affinity (Kd ≤ 1 nM), and this binding survives the competition from other ribosome-associated protein biogenesis factors such as SRP and trigger factor (TF). The RodZ TMD is flanked by basic residues at the N-terminus and acidic residues at the C-terminus (net charge of -4), both of which are important for high affinity binding of SecA in preference over SRP (Wang et al., 2017).
However, little is known about how SecA protects hydrophobic TMDs emerging from the ribosome exit tunnel, nor the molecular basis of its charge preferences during this recognition.
SecA was known to be an essential ATPase that drives the post-translational translocation of secretory proteins harboring less hydrophobic signal sequences across SecYEG(Hartl et al., 1990), often in collaboration with the chaperone SecB. In this post-translational mode, SecA binds the signal sequence via a hydrophobic groove in the pre-protein crosslinking (PPXD) domain (Gelis et al., 2007; Kimura et al., 1991). Another surface on SecA, Patch A, provides additional contact sites for hydrophobic segments in the mature regions of secretory proteins (Chatzi et al., 2017). In its recently described co-translational mode of targeting, it is unclear whether SecA uses the same preprotein binding sites to recognize nascent TMDs emerging from the ribosome. Furthermore, SecA binds with high affinity to anionic phospholipids and to SecYEG (Bauer and Rapoport, 2009; Zimmer et al., 2008), and previous structures (Frauenfeld et al., 2011; Park et al., 2014) suggested that SecA and the ribosome share partially overlapping binding sites on SecYEG. Biochemical data also indicate that SecA and the 70S ribosome
compete for binding to SecYEG (Wu et al., 2012). How the co-translational recognition by SecA leads to the efficient delivery of nascent membrane proteins to SecYEG (Rawat et al., 2015;
Wang et al., 2017) remains an outstanding puzzle.
To address these questions, we combined biochemical and structural analyses to study the molecular mechanism of this pathway. Site-specific crosslinking showed that the ribosome induces a distinct mode of nascent protein recognition by SecA. A cryoEM structure of SecA bound to RNCRodZ showed that the nascent TMD is sandwiched in a composite binding site formed by the N-terminal amphipathic helix of SecA and a hydrophobic groove on uL23 of the ribosome, and revealed the molecular basis for the charge preference during nascent protein
recognition by SecA. Finally, quantitative kinetic analyses demonstrate that SecYEG remodels the RNC-bound SecA to facilitate nascent protein transfer to SecYEG, and the transfer process is further facilitated by the elongation of the nascent polypeptide.